OLIGODENDROGLIAL TROPHIC FACTOR AND METHODS OF USE THEREOF

Abstract: A 60 kilodalton protein ("oligodendroglial trophic factor", OTF) induces the accumulation of oligodendroglia in cultures of cerebral white matter. Half-maximal oligodendroglial recruitment is obtained with 2 ng/mL of oligodendroglial trophic factor. Oligodendroglial trophic factor is a mitogen for oligodendroglial progenitor cells and oligodendroglia, but does not stimulate proliferation of astroglia, Schwann cells, or endoneurial fibroblasts. Immunohistological studies with a monoclonal oligodendroglial trophic factor antibody indicate that oligodendroglial trophic factor is expressed by neurons in CNS and dorsal root ganglia.

Description: OLIGODENDROGLIAL TROPHIC FACTOR AND METHODS OF USE THEREOF FIELD OF THE INVENTION This invention is directed to oligodendroglial trophic factor and methods of use thereof . BACKGROUND OF THE INVENTION Demyelinating diseases, such as multiple sclerosis and perivenous encephalomyelitis are debilitating illnesses. During later stages of multiple sclerosis unsteadiness of gait, incontinence and paralysis due to the widespread cerebral and spinal cord demyelination are common. The condition most often leads to death. Often those suffering from multiple sclerosis suffer a relapsing and remitting course over many years. Perivenous encephalomyelitis is characterized by a monophasic, rapid course which frequently leads death. Demyelinating diseases have in common as the major pathological process the loss of the myelin sheath that surrounds and insulates the axon. Multiple sclerosis has been the subject of exhaustive, yet inconclusive studies. Still, no effective treatment for these diseases is known. Oligodendroglia cells produce myelin that surrounds the axons in the central nervous system. Oligodendroglia are derived from neuroectodermal stem cells of the CNS germinal matrix. Goldman J.E., et al . , J Neurosci 1986, 6, 52-60; LeVine S.M. and Goldman J.E., J Comp Neurol 1988 277, 441- 455. These stem cells give rise to intermediates in the oligodendroglial lineage that are small, process-bearing, motile, and rapidly proliferating. Raff M.C., et al . Na ture 1983, 303 , 390-396; Small R.K., et al . Nature 1987, 328 , 155- 157; LeVine S.M. and Goldman J.E., J " Comp Neurol 1988, 277, 441-455; Noble M.D. et al . , Na ture 1988, 333 , 560-562; Grinspan J.B., et al . , J Neurosci 1990, 10 , 18661-18873; Hardy R. and Reynolds R., Development 1991, 111 , 1061-1070. In the rat, cells at this intermediate stage can be identified immunohistologically by use of monoclonal antibodies (mAbs) that recognize ganglioside and other plasma membrane epitopes, and by their lack of expression of antigens characteristic of astroglia, eg., glial fibrillary acidic protein (GFAP) ) or oligodendroglia (eg, galactocerebroside (galC) , myelin basic protein (MBP) , and proteolipid (PLP) . Reviewed by Goldman, 1992. Proliferation, migration, and differentiation of these motile oligodendroglial progenitor cells, and survival of differentiated oligodendroglia, are regulated by paracrine growth factors, including basic fibroblast growth factor (basic FGF) ; McKinnon R.D. , et al. , Neuron 1990, 5, 603-614; Bogler 0., et al. , Proc Natl Acad Sci USA 1990, 87, 6368- 6372; platelet-derived growth factor (PDGF) ; Richardson .D., et al., Cell 1988, 53 , 309-319; Noble M.D., et al . , Nature 1988, 333 , 560-562; Pringle N. et al. , EMBO J 1989, 8, 1049- 1056; Grinspan J.B., et al . , J Neurosci 1990, 10, 18661- 18873; Barres B.A., et al. , Cell 1992, 70, 31-46; and insulin-like growth factor-1 (IGF-1) ; McMorris F.A. and Dubois-Dalcq M. , J Neurosci Res 1988, 21 , 199-209. Some oligodendroglial growth factors having molecular weights ranging from 16-18kDa which have been released to the medium by various neuroblastoma cell lines have been found. Bottenstein J.E., et al. , J " Neurosci Res, 1988, 20, 291-303; Hunter S.F. and Bottenstein J.E., J Neurosci Res, 1991, 28 : 574-582; Giulian D., et al . , J Neurosci 1991, 11 , 327-336. The molecular characteristics of the mitogens for cells of the oligodendroglial lineage released to the medium by primary cultures of CNS neurons; LeVine M.L., Neuron 1989, 3, 103-113; Gard A.L. and Pfeiffer S.E., Neuron 1990, 5, 615-625; have not yet been reported. Tissue culture studies indicate that oligodendroglial differentiation can occur in the absence of neurons. Raff M.C., et al . , Na ture 1978, 274 , 813-816; Barbarese E. and Pfeiffer S.E., Proc Natl Acad Sci USA 1981, 78 , 1953-1957; Zeller N.K., et al . , J Neurosci 1985, 5, 2955- 2962; Saneto R.P. and De Vellis J. , Proc Na tl Acad Sci USA 1985, 82 , 3509-3513; Knapp P.E., et al. , Dev Biol 1987, 120, 356-365; Grinspan J.B., et al . , J Neurosci 1990, 10 , 18661- 18873; Hardy R. and Reynolds R. , Development 1991, 111 , 1061- 1070. However, oligodendroglia are able to assemble and maintain compact myelin only if in contact with axons of appropriate caliber. Wood P.M. and Williams A.K., Dev Brain Res 1984, 12, 225-241; Kidd G.J., et al . , J Neurosci Res 1990, 26, 409-418. This contact is stabilized by adhesive interaction between oligodendroglial MAG and an as yet uncharacterized neuronal plasma membrane constituent . Johnson P.W., et al . Neuron 1989, 3, 377-385; Owens G.C., et al . , J Cell Biol 1990, 111 , 1171-1182; Quarles R.H., et al . , In: Myelin : Biology and Chemistry (Martenson RE, ed) , pp 413- 448. Boca-Raton: CRC Press (1992) . A second contact- mediated oligodendroglial-neuronal interaction is the inhibition of axonal elongation induced by oligodendroglial membrane-bound surface proteins. Caroni P. and Schwab M.E., Dev Biol 1989, 136, 287-295; Fawcett J.W. , et al. , J Cell Sci 1989, 92, 93-100. Several observations suggest the existence of yet a third contact-dependent trophic interaction between cells of the oligodendroglial lineage and neurons, the induction of oligodendroglial mitosis. Oligodendroglia co-cultivated with dorsal root ganglion neurons proliferate; Wood P.M. and Bunge R.P., Nature 1986, 320 756-758; Wood P.M. and Bunge R.P., Glia 1991, 4 , 225-232; as do oligodendroglia cultured with an axolemma-enriched membrane fraction; Chen S.J. and DeVries G.H., J Neurochem 1989, 52 , 325-327; Vick R.S. and DeVries G.H., J Neurosci Res 1992, 33 , 68-74. The nature of the axolem a-associated oligodendroglial mitogen has not yet been established, although one study suggests it is acidic fibroblast growth factor (acidic FGF) or a related protein; Vick R.S. and DeVries G.H., J Neurosci Res 1992, 33 , 68-74, and another that is a novel, 50 kDal extrinsic axonal membrane protein having a half maximal activity of 200 ng/ML; Nordlund M. , et al . , Glia 1992, 5, 182-192. This 50kDa protein is also a Schwann cell mitogen. The induction of oligodendroglial mitosis to replace myelin lost during the course of multiple sclerosis and other demyelinating diseases is greatly desired. SUMMARY OF THE INVENTION Applicants have identified and isolated a novel oligodendroglial trophic factor. This protein has been found by Applicants to induce mitosis of oligodendroglia, thereby inducing replacement of myelin lost during the progression of demyelinating diseases such as multiple sclerosis and perivenous encephalomyelitis. In accordance with one preferred embodiment of the present invention, methods of isolating an oligodendroglial trophic factor comprising preparing a 0.5M salt extract of mammalian whole brain, incubating the extract with an ion exchange matrix at pH 7.4 in 0.15M NaCl,running the unbound supernatant on a size exclusion column having a fractionation range of from 5kDa to 600kDa, eluting fractions, collecting fractions having oligodendroglial trophic activity, and resolving the active fractions on an denaturing gel, are provided. In accordance with another preferred embodiment of the present invention is provided methods of inducing mitosis of oligodendroglia comprising the steps of preparing a 0.5M salt extract of mammalian whole brain, incubating the extract with an ion exchange matrix at pH 7.4 in 0.15M NaCl, running the unbound supernatant on a size exclusion column having a fractionation range of from 5kDa to 600kDa, eluting fractions, collecting fractions having oligodendroglial trophic activity, resolving the active fractions on an denaturing gel, recovering a protein having an approximate molecular weight of 60kDa from the gel, and contacting oligodendroglia with an amount of recovered protein effective to induce mitosis of the contacted oligodendroglia. In accordance with further a preferred embodiment of the present invention are provided methods of treating a demyelinating disease in a patient suffering from a demyelinating disease comprising the steps of preparing a 0.5M salt extract of mammalian whole brain, incubating the extract with an ion exchange matrix at pH 7.4 in 0.15M NaCl, running the unbound supernatant on a size exclusion column having a fractionation range of from 5kDa to 600kDa, eluting fractions, collecting fractions having oligodendroglial trophic activity, resolving the active fractions on an denaturing gel, recovering a protein having an approximate molecular weight of 60 kDa from the gel, and administering to the patient suffering from a demyelinating disease an amount of recovered protein effective to induce mitosis of oligodendroglia in said patient. In still a further preferred embodiment of the present invention, substantially pure oligodendroglial trophic factor comprising a protein having a molecule weight of approximately 60 kD said protein exhibiting an oligodendroglial trophic activity of about 400 units/μg is provided. Further in accordance with a preferred embodiment of this invention is provided a pharmaceutical composition comprising oligodendroglial trophic factor in a pharmaceutically acceptable carrier. In yet a further preferred embodiment of this invention is provided recombinant host cells transformed with a portion of DNA coding for oligodendroglial trophic factor sufficient for the expression of oligodendroglial trophic factor by said host cells. Another preferred embodiment of the present invention provides methods of producing oligodendroglial trophic factor comprising culturing recombinant host cells wherein a recombinant DNA transformed in said host cell has a DNA sequence encoding oligodendroglial trophic factor, operably linked to appropriate regulatory control sequences which are capable of effecting the expression of said coding sequences in said transformed cells. Recombinant vectors capable of expression in a suitable expression system which comprises a DNA sequence encoding oligodendroglial trophic factor operably linked to control sequences compatible with said expression system are also provided by a preferred embodiment of this invention. Further, a preferred embodiment of the present invention provides hybridoma cells lines which express monoclonal antibodies which specifically recognize oligodendroglial trophic factor. A preferred embodiment of the present invention also provides methods of inducing mitosis of oligodendroglia comprising contacting oligodendroglia with an amount of oligodendroglial trophic factor effective to induce mitosis of oligodendroglia. In a further preferred embodiment of the present invention is provided methods of treating demyelinating diseases comprising administering to a patient suffering from a demyelinating disease an amount of oligodendroglial trophic factor effective to induce mitosis of oligodendroglia of the central nervous system. Applicants have identified an oligodendroglial trophic factor which induces mitosis of oligodendroglia. The oligodendroglial trophic factor is isolated herein from mammalian whole brain based upon its oligodendroglial trophic activity. BRIEF DESCRIPTION OF THE FIGURES Figure 1 sets forth photographs of the results of the treatment of neonatal rat CWM cultures with NaCl extract of brain membranes. Indirect immunofluorescence microscopy was used to identify A2B5 + cells (panels A and C) or R-mAb + cells (panels B and D) in neonatal rat CWM cultures. Panels C and D shown the appearance of a culture maintained in medium to which NaCl extract of brain membranes (20 μg/mL) had been added 3 days prior to microscopy, panels A and B the appearance of a parallel, untreated culture. The size bar = 36 μm. Figure 2 is a photograph showing the effects of NaCl extract of brain membranes on the synthesis of DNA by a CWM culture. A CWM culture was treated for 2 days with 20 μg/mL of NaCl extract of brain membranes, and incubated with BrdU (lOμM) for the second of these 2 days. Panel A shows 2 oligodendroglia that bind R-mAb to their plasma membranes, and panel B shows that, after fixation these two oligodendroglia bind an anti-BrdU mAb to their nuclei. Similar results were obtained when the 01 mAb was substituted for the R-mAb in such studies (now shown) . Figure 3 is a schematic representation comparing the rates of DNA synthesis of oligodendroglia and 0-2A cells following the addition of NaCl extract of brain membranes. NaCl extract of brain membranes (20 μg of protein/mL) was added at day 0 (4 days post-isolation of the CWM cells) and again with refeeding of fresh medium on days 3 and 7. BrdU was added to the medium 24 hours (for the oligodendroglial studies, panel A) or 6 hours (for the 0-2A cell studies, panel B) prior to examination of the cultures by dual wavelength indirect immunofluorescence microscopy. Oligodendroglia were identified by surface binding of the R- mAb. 0-2A cells were identified by surface binding of the A2b5 mAb. Data are the means of 4 independent experiments, with vertical bars denoting SEMs. White bars = untreated cultures; black bars = cultures treated with NaCl extract. Figure 4 is a reproduction of a silver stained SDS- PAGE gel. Lane 1 of the silver-stained gel was loaded with 2 μg of post-S200 Sephacryl oligodendroglial trophic factor, lane 2 with 10 μg of post-DEAE oligodendroglial trophic factor. Elution of proteins from a parallel, non-stained lane of post-S200 Sephacryl oligodendroglial trophic factor (see Methods) indicated that all galC + cell recruiting activity was recovered in the region of the prominent, 60 kDal (MW app ) band. Figure 5 is an image of a MAG immunofluorescence microscopy of a CWM culture treated with oligodendroglial trophic factor. This CWM culture was treated with post-S200 Sephacryl oligodendroglial trophic factor (lOOng/mL) for 10 days, then examined by dual wavelength indirect immunofluorescence microscopy for binding of the R-mAb (Panel A) and anti-MAG immunoglobulins (Panel B) . The size-bar = 33 micrometers. Figure 6 is an image of a MBP immunofluorescence microscopy of CWM cultures treated with oligodendroglial trophic factor or PDGF. Panels A and C show the phase- contrast and MBP indirect immunofluorescence appearances of a field from a CWM culture treated for 10 days with human PDGF (2 ng/mL) ; about half of the oligodendroglia in the field are MBP + . Panels B and D show the phase-contrast and MBP indirect immunofluorescence appearances of a field from a CWM culture treated for 10 days with post-S200 Sephacryl oligodendroglial trophic factor (100 ng/mL) . Only a few MBP + cells are present. The size bar = 26 micrometers. Figure 7 is an autoradiogram of a Northern blot analysis of MBP mRNA in CWM cultures treated with oligodendroglial trophic factor, PDGF, or basic FGF. CWM cultures were treated for 7 days with PDGF (2ng/mL) , basic FGF (10 ng/mL) , or post-S200 Sephacryl oligodendroglial trophic factor (lOOng/mL) . Total RNA (10 μg/lane for basic FGF and oligodendroglial trophic factor treated cultures, 5 μg/lane for PDGF treated culture) was examined by Northern blotting with an MBP cDNA probe. The upper panel shows the autoradiogram obtained with a [ 32 P] -labelled MBP cDNA probe. The MBP mRNA signal is far less intense in the basic FGF and oligodendroglial trophic factor lanes than in the PDGF lane. Rehybridization of the blot with a [ 32 P] -labelled PLP cDNA probe demonstrated that the signal in the PDGF lane is far more intense than in the basic FGF and oligodendroglial trophic factor lanes (results not shown) . The lower panel illustrates the methylene blue-stained 28S bands in the same 3 lanes, showing that amounts of RNA loaded on the bFGF and oligodendroglial trophic factor lanes were similar, while loading of the PDGF lane was less. Figure 8 is two panels of images showing the immunohistological localization of oligodendroglial trophic factor. Panel A shows Paraffin sections of human cerebellum labelled with peroxidase after incubation with the oligodendroglial trophic factor mAb, Du; the section was counterstained with hematoxylin. Panel B shows P6 dorsal root ganglion cells labelled with peroxidase after incubation with Du mAb; the section was viewed with Zeiss differential interference contrast optics. DETAILED DESCRIPTION OF THE INVENTION In accordance with some embodiments of the present invention is provided substantially pure oligodendroglial trophic factor (OTF) . The term "oligodendroglial trophic factor" refers to a protein having oligodendroglial trophic activity. By oligodendroglial trophic activity is meant the induction of mitosis of oligodendroglia cells above background levels. Mitosis, of course, is well understood by those skilled in the art to mean the process whereby chromosomes of a cell duplicate and segregate, followed by division of the cell into two daughter cells, each containing identical sets of chromosomes. Preferably, oligodendroglial trophic activity is referred to in terms of the number of additional oligodendroglia/mττv 2 over background resulting after the addition of oligodendroglial trophic factor. One unit of activity is defined as being equal to the addition of ten oligodendroglia cells/mm 2 over background. While oligodendroglial trophic factor stimulates the mitosis of oligodendroglia, it does not stimulate the production by these oligodendroglia of myelin structural proteins such as MPB or PLP, proteins which are largely responsible for stabilizing the interactions between the lipid bilayers within compact myelin, thereby facilitating the initial ensheathment of axons by these oligodendroglia. Oligodendroglial trophic factor is neuron associated. Oligodendroglial trophic factor may be isolated from a whole brain extract by a combination of ionic and size separations followed by resolution on an SDS polyacrylamide gel . Accumulation of oligodendroglia in rat cerebral white matter cultures can be used as an assay to monitor the course of purification of oligodendroglial trophic factor from a 0.5M NaCl membrane extract. Thereafter, the. extract may be incubated with an ion exchange matrix such as DEAE Sephadex at pH 7.4 in 0.15M NaCl. The unbound supernatant can be run on a size exclusion column having a fractionation range of from 5KDa to about 600KDa. Fractions may be eluted and the fractions having oligodendroglial trophic activity collected. Proteins in the active fractions can be resolved using a denaturing gel. Bioactive oligodendroglial trophic factor did not bind to DEAE Sephadex at pH 7.4 in 0.15M NaCl, and was eluted from a S-200 Sephacryl column, in a fraction corresponding to a molecular weight, for globular proteins, of 50-70 kDa. Resolution on SDS polyacrylamide gel indicated that the post- S-200 Sephacryl preparation contained a major protein band with an apparent molecular weight under reducing conditions of approximately 60kDa. Oligodendroglial trophic factor activity was recovered by elution of the 60kDa band. Half- maximal accumulation of R-mAb + and of 01 + oligodendroglia in cerebral white matter cultures was obtained with a concentration of 2ng of post-SDS-PAGE oligodendroglial trophic factor protein/mL. Thus, overall, oligodendroglial trophic factor was purified 4,000 fold over the original NaCl extract to substantial purity. In some embodiments of the present invention "substantially pure" means purity of greater than about 85%. In other embodiments of the present invention purity of greater than 90% is preferred. Most preferred is greater than 95% purity. In some embodiments of the present invention the oligodendroglial trophic factor has a molecular weight of ranging from approximately 50kD to approximately 70kD. In most preferred embodiments of the present invention, oligodendroglial trophic factor has a molecular weight of 60kDa. Precursors of the oligodendroglial trophic factor are also encompassed herein, as are biologically active fragments of the oligodendroglial trophic factor. The oligodendroglial trophic factor of the present invention, depending on the pH of its environment, if suspended or in solution, or of its environment when crystallized or precipitated, if in solid form, may be in the form of pharmaceutically acceptable salts or may be in neutral form. The free amino groups of the protein are, of course, capable of forming acid addition salts with, for example, organic acids such as hydrochloric, phosphoric, or sulfuric acid; or with organic acids such as, for example, acetic, glycolic, succinic, or mandelic acid. The free carboxyl groups are capable of forming salts with bases, including inorganic bases such as sodium, potassium, or calcium hydroxides, and such organic bases as piperidine, glycosamine, trimethylamine, choline, and caffeine. In addition, the protein may be modified by combination with other biological materials such as lipids and saccharides, or by side chain modification such as acetylation or amino groups, phosphorylation of hydroxyl side chains, or oxidation of sulfhydryl groups. Modifications of oligodendroglial trophic factor are included within the scope of the definition, so long as the oligodendroglial trophic activity as described herein is retained. Finally it is understood that minor modifications of oligodendroglial trophic factor may result in proteins which have substantially equivalent or enhanced trophic activity. These modifications may be deliberate, as through site directed mutagenesis, or may be accidental, such as through mutation in hosts which are oligodendroglial trophic factor producers. All of these modifications are included as long as the trophic activity is retained. Oligodendroglial trophic factor of the present invention may be naturally occurring or may be recombinantly produced. Oligodendroglial trophic factor can be prepared directly from mammalian whole brain as described. The oligodendroglial trophic factor may further be characterized by amino acid sequence and compositional analysis by standard techniques known in the art such as for example, amino acid analysis using an amino acid analyzer with automated hydrolysis (Applied Biosystems model 420, ABS, Foster City, CA) . Sequence analysis can be performed by the Ed an degradation method using for example, a pulsed liquid phase sequencer (Applied Biosystems model 477'A, ABS) . Cysteine residues may be identified by sulfhydryl reduction following by reaction with 4-vinylpyridine as described by Selsted, M.E., S.S. Harwig, et al. , J. Clin . Inv. , 1985, 76 (4) : 1436- 1439, prior to sequence analysis. Mass spectra analysis may be used to determine the molecular weight of the peptide such as by fast atom bombardment. It is further believed that polypeptides can be routinely synthesized in substantially pure form by standard techniques well known in the art, such as, for example, commercially available peptide synthesizers. The oligodendroglial trophic factor may also be prepared recombinantly. For example, a cDNA expression library can be used to isolate the DNA sequence coding for oligodendroglial trophic factor. cDNA libraries are commercially available or may be prepared in accordance with methods known in the art. Monoclonal antibody to oligodendroglial trophic factor can be used to screen the library and plaques which are recognized by the antibody can be sequenced. Monoclonal antibodies which specifically recognize oligodendroglial trophic factor are provided by the present invention as are hybridoma cell lines which may be used to produce such monoclonal antibodies. By specifically recognized is meant that oligodendroglial trophic factor is bound by the antibody, to the exclusion of other proteins which may be present . Alternatively a DNA library may be screened using a nucleic acid probe which specifically recognizes the oligodendroglial trophic factor gene. Such nucleic acid probe may be prepared based upon the amino acid sequence of the oligodendroglial trophic factor. Nucleic acid sequences coding for all or portions of oligodendroglial trophic factor are encompassed by some embodiments of the present invention. Based upon the determined nucleic acid sequence it is believed the oligodendroglial trophic factor or fragments thereof may be prepared using modifications of any of numerous well known recombinant techniques such as those described in U.S. Patent #4,677,063 which patent is incorporated by reference as if fully set forth herein. By "portion thereof" as used herein is meant to refer to any portion of a peptide or nucleic acid of sufficient size to retain desired biological activities, and particularly oligodendroglial trophic activity. Briefly, most of the techniques which are used to transform cells, construct vectors, extract messenger RNA, prepare cDNA libraries, and the like are widely practiced in the art, and most practitioners are familiar with standard resource materials which describe specific conditions and procedures. However, for convenience, the following paragraphs may serve as a guideline. Procaryotes most frequently are represented by various strains of E. coli . However, other microbial strains may also be used, such as Bacilli , for example, Bacillus s btilis, various species of Pseudomonas , or other bacterial strains. In such procaryotic systems, plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used. In one such "expression system" for example, E. coli is transformed using a derivative of pBR322, a plasmid derived from an E. coli species by Bolivar, et al . , Gene 2 : 95 (1977) . pBR322 contains genes for ampicillin and tetracycline resistance and thus provide additional markers which can be either retained or destroyed in constructing the desired vector. Commonly used procaryotic control sequences include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al . , Nature 198 : 1056 (1977) ) and the tryptophan (trp) promoter system (Goeddel, et al . , Nucleic Acids Res 8 . : 4057 (1980)) and the lambda derived P L promoter and Ν-gene ribosome binding site (Shimatoake, et al. , Nature 292 : 128 (1981)) . In addition to bacteria, eucaryotic microbes, such as yeast may also be used as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker's yeast, are most used although a number of other strains are commonly available. While vectors employing the 2 micron origin of replication are illustrated, Broach, J.R., Meth Enz . 101: 307 (1983) , other plasmid vectors suitable for yeast expression are known (see, for example, Steinchcomb, et al. , Nature 282 : 39 (1979) , Tschempe, et al . , Gene 10: 157 (1980) and Clark, L., et al., Meth Enz 101: 300 (1983)) . Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess, et al . , J. Adv Enzyme Req 2= 14 9 (1968) ; Holland, et al . Biochemistry 17: 4900 (1978)) . Additional promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeman, et al . , J " . Biol . Chem 255 : 2073 (1980) ) , and those for other glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization (Holland, ibid) . It is also believed terminator sequences are ' desirable at the 3' end of the coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes. Many of the vectors illustrated contain control sequences derived from the enolase gene containing plasmid peno46 (Holland, M.J., et al. , J. Biol Chem 256 : 1385 (1981)) or the LEU2 gene obtained from YEpl3 (Broach, J., et al., Gene 8.: 121 (1978)), however any vector containing a yeast compatible promoter, origin of replication and other control sequences is suitable. It is also of course possible to express genes encoding polypeptides in eucaryotic host cell cultures derived from multicellular organisms. See, for example, Tissue Cul tures, Academic Press, Cruz and Patterson, editors (1973) . Useful host cell lines include VERO, HeLa cells, and Chinese hamster ovary (CHO) cells. In vivo expression of genes encoding polypeptides in eucaryotic hosts is also a feasible method of preparation of gastrointestinal defensin peptides . Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from Simian Virus 40 (SV 40) Fiers, et al., Nature 273 : 113 (1978)), or other viral promoters such as those derived from polyoma, Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses. General aspects of mammalian cell host system transformations have been described e.g. by Axel; U.S. pat. No. 4,399,216. It now appears, also that "enhancer" regions are important in optimizing expression; these are, generally, sequences found upstream or downstream of the promoter region in non-coding DNA regions. Origins of replication may be obtained, if needed, from viral sources. However, integration into the chromosome is a common mechanism for DNA replication in eucaryotes. Plant cells are now available as hosts, and control sequence compatible with plant cells such as the nopaline synthase promoter and polyadenylation signal sequences (Depicker, A., et al. , J. Mol Appl . Gen 1 : 561 (1982)) are available. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described by Cohen, .N. , Proc Natl Acad Sci (USA) 9_: 2110 (1972) , or methods described in Molecular Cloning: A Laboratory Manual (1988) Cold Springs Harbor Press, could be used for procaryotes or other cells which contain substantial cell wall barriers. Infection with Agrobacterium tumefaciens (Shaw, CH., et al . , Gene 23 . : 315 (1983)) is believed useful for certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology .52.: 546 (1978) can be used. Cells may also be transformed in vivo by introducing a gene, incorporated in a vector, directly to cells in the walls of blood vessels using, for example, balloon catheter techniques. WO 90/11734 issued October 18, 1990. Transformations into yeast can be carried out according to the method of Van Solingen, P., et al. , J " Bact 130: 946 (1977) and Hsiao, C.L. , et al . , Proc Natl Acad Sci (USA) 76.: 3829 (1979) . cDNA or genomic libraries can be screened using the colony hybridization procedure. Generally, each microtiter plate is replicated onto duplicate nitrocellulose filter papers (S&S type BA-85) and colonies are allowed to grow at 37°C for 14-16 hr on L agar containing 50 μg/ml Amp. The colonies are lysed and DNA fixed to the filter by sequential treatment for 5 min with 500 mM NaOH, 1.5M NaCl, and are washed twice for 5 min each time with 5X standard saline citrate (SSC) . Filters are air dried and baked at 80°C for 2 hr. The duplicate filters are prehybridized at 42°C for 6-8 hr with 10 ml per filter of DNA hybridization buffer (5xSSC, pH 7.0 5X Denhardt's solution (polyvinylpyrrolidine, plus Ficoll and bovine serum albumin; lx=0.02% of each) , 50 mM sodium phosphate buffer at pH 7.0, 0.02% SDS, 20 μg/ml Poly U, and 50 μg/ml denatured salmon sperm DNA) . The samples can be hybridized with kinased probe under conditions which depend on the stringency desired. Typical moderately stringent conditions employ a temperature of 42° C for 24-36 hr with 1-5 ml/filter of DNA hybridization buffer containing probe. For higher stringencies high temperatures and shorter times are employed. Generally, the filters are washed four times for 30 min each time at 37°C with 2xSSC, 0.2% SDS and 50 mM sodium phosphate buffer at pH 7, then are washed twice with 2xSSC and 0.2% SDS, air dried, and are autoradiographed at -70° C for 2 to 3 days. Construction of suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired. Site specific DNA cleavage can be performed by treating the DNA with a suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, about 1 μg of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 μl of buffer solution. Incubation times of about one hour to two hours at about 37-? C are workable, although variations can be tolerated. After each incubation, protein can be removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol followed by running over a Sephadex G-5 spin column. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations can be found in Methods in Enzymology (1980) 65: 499-560. Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20° to 25° C in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present. If desired, selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the sticky ends. After treatment with Klenow, the mixture is extracted with phenol/chloroform and ethanol precipitated followed by running over a Sephadex G-50 spin column. Treatment under appropriate conditions with SI nuclease results in hydrolysis of any single-stranded portion. Synthetic oligonucleotides can be prepared by the triester method of Metteucci, et al . J " Am Chem Soc 103 : 3185 (1981) ) or using commercially available automated oligonucleotide synthesizers. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 0.1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl 2 , 5 mM dithiothreitol, 1-2 Mm ATP, 1.7 pmoles γ 32 P- ATP (2.9 mCi/mmole) , 0.1 mM spermidine, 0.1 mM EDTA. Ligations can be performed in 15-30 μl volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 μg/ml GSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C (for "blunt end" ligation) . Intermolecular "sticky end" ligations are usually performed at 33-100 μg/ml total DNA concentrations (5-100 mM total end concentration) . Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 μM total ends concentration. In vector construction employing "vector fragments", the vector fragment can be treated with bacterial alkaline phosphatase (BAP) in order to remove the 5' phosphate and prevent religation of the vector. BAP digestions can be conducted at pH 8 in approximately 150 mM Tris, in the presence of Na+ and Mg+ 2 using about 1 unit of BAP per μg of vector at 60° C for about one hour. In order to recover the nucleic acid fragments, the preparation is extracted with phenol/chloroform and ethanol precipitated and desalted by application to a Sephadex G-50 spin column. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments. For portions of vectors derived from cDNA or genomic DNA which require sequence modifications, site specific primer directed mutagenesis can be used. This is conducted using a primer synthetic oligonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage. Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. The resulting plaques can be hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered. Correct ligations for plasmid construction can be confirmed by first transforming a suitable host with the ligation mixture. Successful transformants are selected by ampicillin, tetracycline or other antibiotic resistance or using other markers depending on the mode of plasmid construction, as is understood in the art. Plasmids from the transformants can then be prepared according to the method of Clewell, D.B., et al . Proc Na tl Acad Sci (USA) G2_ : 1159 (1969) , optionally following chloramphenicol amplification (Clewell, D.B., J. Bacteriol 110: 6670 (1972) . The isolated DNA is analyzed by restriction and/or sequenced by the dideoxy method of Sanger, F., et al . Proc Natl Acad Sci (USA) I : 5463 (1977) as further described by Messing, et al . , F. Supp. Nucleic Acids Res 9_: 309 (1981) , or by the method of Maxam, et al . , Methods in Enzymology 65 . : 499 (1980) . Also provided by this invention are methods of inducing differentiation of oligodendroglia comprising contacting the oligodendroglia with an effective amount of oligodendroglial trophic factor. It has been found that ng quantities of oligodendroglial trophic factor are effective to induce mitosis in oligodendroglia. In accordance with still other aspects of the invention are methods of treating patients suffering from a demyelinating disease comprising administering to a patient suffering from a demyelinating disease an effective amount of oligodendroglial trophic factor. One skilled in the art would be familiar with methods of administration which may be effective for treatment of demyelinating disease in view of the present disclosure. For example administration may be carried out intravascularly, orally, rectally, etc., using a variety of dosage forms. Treatment is performed until the desired therapeutic result is achieved. One skilled in the art will recognize the particular mode of administration, dosage, and duration will vary depending upon the age, weight and condition of the patient. Oligodendroglial trophic factor may be formulated into pharmacological compositions containing an effective amount of oligodendroglial trophic factor and a usual nontoxic carrier, such carriers being known to those skilled in the art. The composition can be given via a route of administration suited to the form of the composition. Such compositions are, for example, in the form of usual liquid preparations including solutions, suspensions, emulsions and the like which can be given orally, intravenously, subcutaneously or intramuscularly. Additionally, for treatment of demyelinating diseases, endogenous expression of oligodendroglial trophic factor may be induced in cells. For example selected for expression of oligodendroglial trophic factor may be transformed in vi tro or in vivo with DNA comprising a portion of an oligodendroglial trophic factor cDNA or genomic sequence. Cells which are transformed in vitro may be introduced into a mammal by methods familiar to those skilled in the art such as by performance of cellular grafts . In vivo transformation may also be accomplished by introduction of a recombinant vector described previously. WO 90/11734 issued October 18, 1990. The following examples are illustrative but are not meant to be limiting of the invention. EXAMPLES EXAMPLE 1 CELL CULTURES Cerebral white matter (CWM) cultures were established from the corpus callosum and surrounding white matter of 6 day postnatal Sprague-Dawley rats and maintained in serum-free medium as described previously. Grinspan J.B., et al., J Neurosci 1990, 10, 18661-18873. Cells were treated with oligodendroglial trophic factor beginning four days after initiation of culture. The medium (with or without added oligodendroglial trophic factor) was replaced at three to four day intervals. In some experiments (indicated in the text and figures) , human platelet-derived PDGF (largely AB heterodimeric; Bowen-Pope D.F., et al . , J Biol Chem 1989, 264 , 2502-2508; was added biweekly at 2 ng/mL with 1% platelet poor human plasma; Grinspan J.B., et al . , J Neurosci 1990, 10, 18661-18873. In others, 10 ng/mL of human recombinant acidic or basic FGF (R&D Systems) was added at 48 hour intervals. Monolayer cultures of neonatal rat sciatic nerve Schwann cells or dorsal root ganglia were established as previously described in Wood P.M. and Williams A.K., Dev Brain Res 1984, 12, 225-241; and Kreider B.Q., et al . , Brain Res 1981, 207, 433-444. EXAMPLE 2 INDIRECT IMMUNOFLUORESCENCE MICROSCOPY Monoclonal antibodies (mAb) against glycolipids (A2B5 (plasma membrane antigen of neurons) , Eisenbarth, et al., Proc . Natl . Acad. Sci . U. S.A . 1979, 76, 4913-4917; anti- GD3 (Goldman J.E., et al . , J Neurosci 1986, 6, 52-60, 01 anti-galactocerebroside (oligodendrocytes cell surface) ; Sommer J. and Schachner M., Dev Biol 1980, 83 , 311-327; and R-mAb (galactocerebroside) ; Ranscht B., et al . , Proc Natl Acad Sci USA 1982, 79, 2709-2713; Bansal R. , et al . , J " Neurosci Res 1989, 24 , 548-557, or a rabbit antiserum raised against the extracellular domain of myelin-associated glycoproteins; Pedraza L., et al . , J Cell Biol 1990, 111 , 2651-2751; used at a dilution of 1:100, and kindly provided by Dr. James Salzer, NYU Medical Center) , were applied to unfixed cultures for 25 min at 4°C (A2B5, 01, R-mAb) or 25°C (anti-MAG) . After washing with Hank's buffered salt solution, and incubation with the appropriate fluorescein- rhodamine-, or Texas red-conjugated secondary antiserum (from Nordic, Organon Teknika, or Jackson Labs) , the cells were fixed with acid-alcohol (95% ethanol, 5% acetic acid, v/v) at -20°C for 10 min. An anti-MBP mAb; Hickey, et al . , J. Histochem . Cytochem . , 1983, 31 , 1126-1135; was applied after acetone fixation, an anti-GFAB (gel-excised glial filament protein) mAb; Lee VM-Y, et al . , J N euro chem 1984, 42 , 25-32; after acid-alcohol fixation. The number of cells binding the various antibodies were determined by examining twenty-one 63x oil-immersion fields equivalent to 1 mm 2 of coverslip surface area. Fields were selected at random and judged to be of appropriate density under phase contrast prior to scanning under epifluorescence illumination. Mitotic indices were estimated by incubating cultures with the thymidine analogue, bromodeoxyuridine (BrdU, lOuM) for the last 4 or 24 hours prior to fixation in acid-alcohol. Indirect immunofluorescence microscopy employing an anti-BrdU (Amersham) was used to determine the proportion of cells of a given phenotype that had incorporated BrdU into their nuclei. Grinspan J.B., et al. J Neurosci 1990, 20, 18661-18873. EXAMPLE 3 Northern blotting Steady-state levels of mRNAs encoding MBP, PLP, or MAG were estimated in CWM cultures maintained with or without oligodendroglial trophic factor, PDGF, or basic FGF by Northern blotting. Sambrook J, Fritsch EF, Maniatis T Molecular Cloning. A Laboratory Manual . 2nd ed, Cold Spring Laboratory Press, 1989. For this purpose, total RNA was isolated from the CWM cultures by the method of Chomcyznski and Sacci, Anal . Biochem. , 1987, 162, 156-159, resolved by electrophoresis on an agarose-formaldehyde gel, and transferred to Duralon membranes (Stratagene) . Prehybridization was for 2 hours at 42°C in 50% formamide, 5 x SSPE (1 x = 0.15M NaCl, 0.01M sodium phosphate and 1 mM EDTA) , 1% SDS, 5 x Denhardt' s solution (1 x - 0.02% Ficoll, 0.02% polyvmylpyrrolidine, 0.02% bovine serum albumin, 100 μg/mL denatured salmon sperm DNA) . Hybridization with full length rat MBP or rat PLP cDNAs; deFerra F., et al . , Cell 1985, 43 , 721-727; Grinspan J., et al . , J " Neurocy tol , in press (1992) ; Kamholz J. , et al . , J Neurosci Res 1992, 31 , 231-244; or with a rat MAG cDNA (a gift from Dr. J. Salzer, NYU Medical Center) , was for 24 hours in the same buffer. The blots were washed once with 2 x SSC at room temperature, once with 2 x SSC at 65°C, and once with 0.2 x SSC at 65°C, and were exposed to Kodak XAR5 film at -70°C. EXAMPLE 4 PREPARATION OF OLIGODENDROGLIAL TROPHIC FACTOR Whole brains from three day postnatal Sprague- Dawley rats were homogenized at 4°C in a solution ("homogenization buffer") containing 0.25 M sucrose, 0.1 mM MgCl 2 , 0.63 mM phenylmethylsulfonylfluoride, and 10 mM Tris (pH 7.0) . The homogenate was centrifuged at 2000 x g for 10 min at 4°C. A soluble extract was prepared from the 130,000 x g pellet by resuspension and incubation in 0.5M NaCl in homogenization buffer for 60 min at 4°C, followed by centrifugation at 130,000 x g for 60 min at 4°C. The supernatant was dialysed overnight at 4°C against phosphate- buffered 150 mM NaCl, pH 7.0 (PBS) ; Ratner N. , et al . , Proc Natl Acad Sci USA 1988, 85, 6992-6996; then stirred overnight with a one-tenth volume of DEAE-Sephadex (Pharmacia) which had been pre-equilibrated with PBS. The suspension was centrifuged. The supernatant was applied to an S-200 Sephacryl column (Pharmacia) , and fractions were eluted with PBS. Those fractions which contained oligodendroglial trophic factor activity were pooled and dialyzed for 16 hours against deionized water at 4°C, lyophilized and stored at - 80°C. Peptides in these fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) ; Laemmli, U.K. Nature 1970, 227, 680-685; and visualized by silver staining. In some experiments, Ponceau- S staining and densitometry were used to estimate amounts of protein in the peptide bands. Segments of unstained SDS-PAGE lanes containing oligodendroglial trophic factor were sliced perpendicular to the direction of migration, washed twice in Ham's F-12 medium at 25°C, each time for 10 minutes, and then proteins were eluted from the slices by shaking overnight at 4°C in Ham's F-12 medium containing 0.1% (w/v) bovine serum 5 albumin. Residual SDS was removed from the supernatants by passing them through small columns of Extracti-gel (Pierce) . Proteins were eluted from these columns with Ham's F-12 medium, and stored at -80°C. Accumulation of oligodendroglia in rat CWM cultures was used as an assay to monitor the 10 course of purification of oligodendroglial trophic factor from the 0.5M NaCl membrane extract as described in Example 5. Bioactive oligodendroglial trophic factor did not bind to DEAE-Sephadex at pH 7.4 in 0.15M NaCl, and was eluted from an S-200 Sephacryl column in a fraction corresponding to a 15 molecular weight (for globular proteins) of 50-70 kDal. Silver staining after SDS-PAGE indicated that the post-S-200 Sephacryl oligodendroglial trophic factor preparation contained a major protein band with an apparent molecular weight under reducing conditions of approximately 60 kDa, 20 together with several fainter bands (Figure 4) . oligodendroglial trophic factor activity was recovered in good yield by elution of the gel segment containing the 60 kDal band. Half-maximal accumulation of R-mAb + and of 01 + oligodendroglia in CWM cultures was obtained with a 25. concentration of 2 ng of post-SDS-PAGE oligodendroglial trophic factor protein/mL. No oligodendroglial trophic factor bioactivity was detected in eluates from other portions of the gel . The overall purification scheme resulted in a 4,000-fold increase in oligodendroglial trophic 30 factor specific activity over that in the original NaCl extract of rat brain membranes, with a yield of 67%. Results are summarized in Table 1. TABLE 1 Purification of oligodendroglial trophic factor Fraction Total Total [Half-max] ** protein activity* NaCl 60 mg 4,800 units 10 μg/mL extract*** Post-DEAE 10 mg 4,000 units 2 μg/mL Sephadex Post-S200 200 μg 3,200 units 50 ng/mL Sephacryl Post-SDS-PAGE 8 μg 3,200 units 2 ng/mL * 1 unit=10 oligodendroglia/mm 2 above the level in untreated cultures ** [Half-max]concentration required for half-maximal recruitment of oligodendroglia *** prepared from brains of 6 liters of 3 day postnatal rats EXAMPLE 5 BIOASSAY OF OLIGODENDROGLIAL TROPHIC FACTOR Oligodendroglial trophic factor activity was assayed by constructing a dose-response curve at each stage of purification to determine the protein concentration that yielded half-maximal recruitment of surface 01 + or R-mAb + oligodendroglia in CWM cultures maintained in defined medium without added PDGF or FGF. Results with the 01 mAb, which binds to galC, and the R-mAb, which binds both to galC and to sulfolipids; Bansal R, et al., J Neurosci Res 1989, 24 , 548- 557; were indistinguishable. One unit of activity was defined as that amount of oligodendroglial trophic factor that yielded 10 additional oligodendroglia per mm 2 . Maximal responses to oligodendroglial trophic factor were about 8 times the untreated background level at each stage of purification. Various antibodies were added to the medium to determine their effects on oligodendroglial trophic factor activity. Rabbit anti-human PDGF (Collaborative Research, 50 μg/mL) was added simultaneously with oligodendroglial trophic factor. Rabbit anti-basic FGF (R&D, 60 μg/mL) was added 90 min before oligodendroglial trophic factor, and rabbit anti- acidic FGF (R&D, 50 μg/mL) was added 30 min before oligodendroglial trophic factor. The anti-oligodendroglial trophic factor mAb, Du (see below) was used as an hybridoma supernatant (1:100,v/v). EXAMPLE 6 OLIGODENDROGLIAL TROPHIC FACTOR MONOCLONAL ANTIBODIES Mice were immunized over the course of 18 days; Brodsky F.M., J Cell Biol 1985, 201, 2047-2054; with post- 3200 Sephacryl oligodendroglial trophic factor, and hybridomas were prepared as described by Arai et al. , Proc . Natl . Acad. Sci . U. S.A . , 1990, 87, 2249-2253. Clones were isolated and their supernatants tested for anti- oligodendroglial trophic factor binding by ELISA; Lee VM-Y, et al., Proceedings of the Natl . Acad. Sci . , USA 1988, 85, 1998-2002; with post-S200 Sephacryl oligodendroglial trophic factor as the target antigen. Two of 6 ELISA-positive clones (Td and Du) blocked oligodendroglial trophic factor-induced accumulation of galC + cells in the CWM cultures. Peroxidase- anti-peroxidase (PAP) immunocytochemistry with Du was used to localize oligodendroglial trophic factor immunoreactivity in tissue sections and dorsal root ganglia (DRG) cultures. Tissue blocks prepared from human cerebellum were fixed with isotonic alcohol and cut into 6 μm paraffin sections. P6 rat DRG cultures were fixed in acid-alcohol, blocked for 30 in with 2% calf serum (v/v) and 0.25% (w/v) cold water fish gelatin in 0.1 M Tris, pH 7.0, then incubated with the oligodendroglial trophic factor mAbs. The paraffin sections and DRG coverslips were then processed for peroxidase-anti- peroxidase immunocytochemistry. Carden (1987) . EXAMPLE 7 PRELIMINARY CHARACTERIZATION OF A CNS MEMBRANE 0.5M NACL EXTRACT Oligodendroglia trophic factor activity was assayed as described in Example 5. The NaCl extract was added at a concentration of 20 μg/mL on days 0 and 3. Numbers of oligodendroglia were determined by counting cells that bound the 01 mAb or R-mAb. Numbers of 0-2A cells were determined by counting cells that bound the A2B5 mAb, but not the 01 mAb or the R-mAb. Results with the 01 mAb and the R-mAb were indistinguishable. Parallel studies with anti-GFAP indicated there was less than 1 type 2 astrocyte/mm 2 in both untreated and treated cultures. Results are the means of 4 independent experiments, with standard errors. Treatment with an 0.5M NaCl extract from crude neonatal rat CNS membranes increased the numbers of cells of the oligodendroglial lineage in neonatal rat CWM cultures (Figure 1 and Table 2) , and also transiently increased the BrdU nuclear labelling indices of such cells (Figures 2 and 3) . TABLE 2 NaCl extract of brain membranes recruits cells of the oligodendroglial lineage in immature rat cerebral white matter cultures Days of Treatment 3 7 Oligodendroglia/mm 2 Control 25 ± 3 27 + 4 NaCl extract 71 ± 9 144 ± 28 0-2A cells/mm 2 Control 20 ± 4 13 ± 2 NaCl extract 93 ± 8 172 ± 36 Numbers and mitotic rates of type 1 astroglia, microglia, and endothelial cells in the rat CWM cultures were not altered by treatment with the 0.5M NaCl extract (data_ not shown) . The NaCl extract-mediated recruitment of oligodendroglia in the CWM cultures was not blocked by antisera that neutralized the actions of basic FGF, acidic FGF, or human PDGF (data not shown) . Increases in the number of oligodendroglia in the CWM cultures induced by the 0.5M NaCl extract and by human PDGF were additive (Table 3) . NaCl extract of brain membrane (20 μg/mL) and human PDGF (2ng/mL) were added on days 0 and 3. Each result is the mean of 4 independent experiments ± standard errors. TABLE 3 NaCl extract of brain membranes and PDGF have additive effects on recruitment of galC + cells in rat CWM cultures Days of Treatment 3 7 GalC + eelIs/mm 2 : Untreated 31 ± 1 30 ± 5 NaCl extract 91 ± 1 193 ± 22 PDGF 28 ± 16 253 + 56 NaCl extract+PDGF 121 ± 57 498 ± 70 Oligodendroglial recruiting activity of the 0.5M NaCl extract did not pass through a dialysis membrane, and was inactivated by trypsin or by heating to 100°C for 5 minutes (data not shown) . Dual label indirect immunofluorescence studies with A2B5 and anti-GFAP; Grinspan J.B., et al., J Neurosci . , 1990, 20, 18661-18873; indicated that less than l type 2 astrocyte/mm 2 ; Raff M.C., et al., Nature 1983, 303, 390-396; was present in both untreated and 0.5M ΝaCl extract-treated CWM cultures (data not shown) . EXAMPLE 9 DETECTION OF OLIGODENDROGLIAL TROPHIC FACTOR STIMULATED DNA SYNTHESIS BY 0-2A OLIGODENDROGLIAL PROGENITORS AND OLIGODENDROGLIA Treatment of CWM cultures with sufficient post-S200 Sephacryl or post-SDS-PAGE oligodendroglial trophic factor t elicit a maximal response, followed 1 day later by a 1 day pulse of BrdU, resulted in a 4- to 5-fold increase in the BrdU labelling indices of both A2B5 + oligodendroglial progenitors and R-mAb * oligodendroglia. Two sets of experiments were designed to establish that oligodendroglial trophic factor exerts a direct 5 mitogenic effect on cells of the oligodendroglial lineage that have reached at least the R-mAb + stage of differentiation, rather than increasing the oligodendroglial BrdU labelling index by enhancing the proportion of rapidly proliferating 0-2A cells that differentiate to 10 oligodendroglia during the BrdU pulse; Fressinaud C. , et al. , J Cell Physiol 1992, 150 , 34-44. In the first set of experiments, the duration of the BrdU pulse was reduced from 24 to 4 hours, thus limiting the time available for maturation of BrdU-labelled 0-2A cells to oligodendroglia. 15 Though the proportion of R-mAb + cells that were BrdU labelled was reduced 5-fold in these experiments, the BrdU labelling index of R-mAb + cells remained 4-fold greater in oligodendroglial trophic factor-treated than in control cultures. The second set of experiments employed cultures 20 that were treated for 3 days with PDGF (2 ng/mL) in order to recruit a cohort of 0-2A cells; Grinspan J.B., et al. J Neurosci 1990, 20, 18661-18873; then maintained for 5 days in the absence of PDGF, during which time most 0-2A cells matured to oligodendroglia. At this point, these cultures 25. contained less than 1 0-2A cell/mm 2 . Treatment of these oligodendrocyte-rich, 0-2A cell-poor cultures with 200 ng/mL of post-S200 Sephacryl oligodendroglial trophic factor elicited a 5-fold stimulation in the R-mAb + cell BrdU labelling index (1 day pulse, initiated 1 day post- 30 oligodendroglial trophic factor addition, n=3) . Results of both sets of experiments thus suggested that oligodendroglial trophic factor exerts a direct mitogenic effect on R-mAb + oligodendroglia. To determine whether oligodendroglial trophic 35 factor is also a mitogen for Schwann cells or endoneurial fibroblasts, sciatic nerve cultures were prepared as previously described; Kreider B.Q., et al. , Brain Res 1981, 207, 433-444; and maintained in a serum-containing or serum- free medium. Crude 0.5M NaCl extract (50 μg/mL) or post-S- 200 Sephacryl oligodendroglial trophic factor (1 μg/mL) was added to the medium, and the cultures were given a 1 day pulse of BrdU 2 days later. In serum-free medium, the crude 0.5M NaCl extract induced a 4-fold increase (n=5) in the BrdU nuclear labelling index of Schwann cells. However, post-S- 200 Sephacryl oligodendroglial trophic factor did not increase the proportion of BrdU-labelled Schwann cells or endoneurial fibroblasts either in serum-free or serum- containing medium (data not shown) . EXAMPLE 10 OLIGODENDROGLIA RECRUITED BY OLIGODENDROGLIAL TROPHIC FACTOR EXPRESS MAG, BUT DO NOT SYNTHESIZE OR EXPRESS MYELIN BASIC PROTEIN (MBP) OR PROTEOLIPID (PLP) About one third of the R-mAb + oligodendroglia in CWM cultures treated with oligodendroglial trophic factor for 3 days or more bound anti-MAG immunoglobulins to their plasma membranes (Figure 5) . Northern blot analysis confirmed the expression of the MAG gene in oligodendroglial trophic factor-treated cultures (data not shown) . Whereas 46% (n=3) of the R-mAb + cells in CWM cultures treated with 2ng/mL of human PDGF for 10 days were MBP + , only 4% (n=3) of R-mAb + cells in 10 day oligodendroglial trophic factor-treated CWM cultures were MBP + (Figure 6) , and this proportion did not increase during an additional 10 days of observation. Northern blot analysis was used to compare steady-state levels of MBP and PLP mRNAs in CWM cultures treated with oligodendroglial trophic factor with those in parallel cultures treated with PDGF or with basic FGF. As would be predicted from the large number of oligodendroglia in PDGF-treated cultures, and their virtual absence from cultures treated with basic FGF, expression of MBP and PLP mRNAs was robust in the PDGF-treated cultures, but barely detectable in the basic FGF-treated cultures (Figure 7) . In the oligodendroglial trophic factor-treated cultures, though cells expressing galactolipids characteristic of oligodendroglia were abundant, MBP and PLP mRNA levels were very low (Figure 7) . This indicates that a pre-translational mechanism is at least in part responsible for the low level of MBP expression by oligodendroglia in oligodendroglial trophic factor-treated CWM cultures. To determine whether oligodendroglial trophic factor suppresses the expression of myelin structural proteins by oligodendroglia in PDGF-treated cultures, CWM cultures were treated simultaneously with oligodendroglial trophic factor and PDGF for 10 days. Indirect immunofluorescence microscopy demonstrated that, as in cultures treated with PDGF alone, about half (49%, n=3) of the R-mAb + cells in these cultures were also MBP + . Previous observations suggested that the synthesis of myelin lipids and myelin structural proteins is tightly coupled during oligodendroglial differentiation. Thus, in cultures maintained in a medium containing basic FGF, cells of the oligodendroglial lineage fail to express both myelin galactosphingolipids (galC and sulfatide) and mRNAs encoding MBP and PLP whereas, in cultures maintained without added basic FGF, mRNAs encoding MBP and PLP appear concurrently with the expression of galC, and immunoreactive MBP and PLP become demonstrable a few days thereafter. Knapp P.E., et al., Dev Biol 1987, 120 , 356-365; Gard A.L. and Pfeiffer S.E., Development 1989, 119-132; Collarini, et al . , Development, 1992, 116, 193-200; Grinspan J., et al. , J Neurocytol , in press (1992) . Contrary to these suggestions, however, in oligodendroglial trophic factor-treated cultures, the coupling between myelin lipid and myelin structural protein synthesis appears to be lost; R-mAb + and 01 + cells accumulate, but fail to express MBP and PLP, though some do express MAG. Conceivably, this failure by oligodendroglial trophic factor to induce expression by new oligodendroglia of PLP and MBP, proteins largely responsible for stabilization of the interactions between the lipid bilayers within compact myelin; Kirschner D.A. and Blaurock A.E., In: Myelin : Biology and Chemistry (Martenson RE, ed) , pp 3-80. Boc- Raton: CRC Press (1992) ; facilitates initial ensheathment of axons by these oligodendroglia. EXAMPLE 11 STUDIES WITH MONOCLONAL ANTIBODY RAISED AGAINST OLIGODENDROGLIAL TROPHIC FACTOR INDICATE OLIGODENDROGLIAL TROPHIC FACTOR IS EXPRESSED BY NEURONS Two of six ELISA-positive oligodendroglial trophic factor mAbs ("Du" and "Te") blocked recruitment of A2B5 + and of R-mAb + cells by oligodendroglial trophic factor in the CWM cultures. For each of the two, half-maximal blocking of the biological activity of 200 ng/mL of post-S200 Sephacryl oligodendroglial trophic factor was obtained with hybridoma supernatants at a dilution of 1:500 (v/v) (data not shown) . Du and Te did not block recruitment of cells of the oligodendroglial lineage induced by PDGF or basic FGF treatment (data not shown) . Immunoperoxidase studies demonstrated that Du bound to the perikarya and axons of Purkinje cells in paraffin sections of human cerebellum, and to a population of large neurons in acid-alcohol fixed cultures of neonatal rat dorsal root ganglia (figure 8) . No binding of Du to fibroblasts or Schwann cells in the rat DRG cultures, or to any of the cell types in the rat CWM cultures, was detected (data not shown) .

Claims: What is claimed is: 1. A method of isolating an oligodendroglial trophic factor comprising: preparing a 0.5M salt extract of mammalian whole brain; incubating the extract with an ion exchange matrix at pH 7.4 in 0.15M NaCl; running the unbound supernatant on a size exclusion column having a fractionation range of from 5kDa to 600kDa; eluting fractions; collecting fractions having oligodendroglial trophic activity; and resolving the proteins in the active fractions on an denaturing gel. 2. An oligodendroglial trophic factor isolated in accordance with the method of claim 1. 3. A method of inducing mitosis of oligodendroglia comprising the steps of: preparing a 0.5M salt extract of mammalian whole brain; incubating the extract with an ion exchange matrix at pH 7.4 in 0.15M NaCl; running the unbound supernatant on a size exclusion column having a f actionation range of from 5kDa to 600kDa; eluting fractions; collecting fractions having oligodendroglial trophic activity; resolving the proteins in the active fractions on an denaturing gel; recovering a protein having an approximate molecular weight of 60kDa from the gel; and contacting oligodendroglia with an effective amount of recovered protein to induce mitosis of the contacted oligodendroglia. 4. A method of treating a demyelinating disease in a patient suffering from a demyelinating disease comprising the steps of : preparing a 0.5M salt extract of mammalian whole brain; incubating the extract with an ion exchange matrix at pH 7.4 in 0.15M NaCl; running the unbound supernatant on a size exclusion column having a fractionation range of from 5kDa to 600kDa; eluting fractions; collecting fractions having oligodendroglial trophic activity; resolving the proteins in the active fractions on an denaturing gel; recovering a protein having an approximate molecular weight from the gel; and administering to the patient suffering from a demyelinating disease an amount of recovered protein effective to induce mitosis of oligodendroglia in said patient. 5. The method of claim 4 wherein the demyelinating disease is multiple sclerosis. 6. A substantially pure oligodendroglial trophic factor comprising a protein having a molecule weight of approximately 60 kD said protein exhibiting an oligodendroglial trophic activity of about 400 units/μg. 7. An oligonucleotide having a sequence coding for the oligodendroglial trophic factor of claim 6. 8. A pharmaceutical composition comprising oligodendroglial trophic factor protein and a pharmaceutically acceptable carrier. 9. Recombinant host cells transformed with a portion of DNA coding for oligodendroglial trophic factor sufficient for the expression of oligodendroglial trophic factor by said host cells. 10. An oligodendroglial trophic factor produced from the recombinant expression vector of claim 9. 11. A method of producing oligodendroglial trophic factor comprising culturing recombinant host cells wherein a recombinant DNA transformed in said host cell has a DNA sequence encoding oligodendroglial trophic factor, operably linked to appropriate regulatory control sequences which are capable of effecting the expression of said coding sequences in said transformed cells. 12. A recombinant vector capable of expression in a suitable expression system which comprises a DNA sequence encoding oligodendroglial trophic factor operably linked to control sequences compatible with said expression system. 13. A hybridoma cell line which expresses monoclonal antibody which specifically recognizes oligodendroglial trophic factor. 14. Monoclonal antibody produced from the hydridoma cell line of claim 13. 15. A method of inducing mitosis of an oligodendroglia comprising contacting the oligodendroglia with an effective amount of oligodendroglial trophic factor. 16. The method of claim 15 wherein from about lOμg/mL to about 2ng/mL oligodendroglial trophic factor is contacted with an oligodendroglia. 17. A method of treating a demyelinating disease comprising administering to said patient suffering from a demyelinating disease with an amount of oligodendroglial trophic factor effective to induce mitosis of oligodendroglia of the central nervous system. 18. The method of claim 17 wherein the demyelinating disease is multiple sclerosis.

Inventors: KREIDER BARBARA Q, LEE VIRGINIA M-Y, PLEASURE DAVID, KREIDER, BARBARA, Q., LEE, VIRGINIA, M-Y, PLEASURE, DAVID
Applicants: PHILADELPHIA CHILDREN HOSPITAL, UNIV PENNSYLVANIA, THE CHILDREN'S HOSPITAL OF PHILADELPHIA, THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA
Application year: 1994
Main classification: C07K3
IPC codes: A61K38, A61P25, C07K1, C07K14, C07K16, C12N5, C12N15, C12P21, C12R1